DESCRIPTION: The goals of our Multi-PI research team are to implement these HTS-ready assays into a full HTS- campaign using the Scripps Institutional Drug Discovery Library to develop, validate and optimize small molecule inhibitors of MITF. Microphthalmia-associated transcription factor MITF is a master regulator of melanocyte differentiation and an oncogene in melanoma, with approximately 20% of melanomas containing high-level MITF. Knockdown of MITF with siRNA induces apoptosis in these MITF-amplified tumors, but also in many melanomas lacking MITF amplification, highlighting it as a putative target for melanoma. However, directly modulating MITF with pharmacologic inhibitors remains challenging, mainly due to the lack of a defined mechanism for inhibition. Our recent research has made breakthroughs that overcame this obstacle. We determined crystal structures of the MITF core region in the apo-form and in the DNA-binding complex, demonstrated that MITF exists as a persistent dimer to function. Surprisingly, we discovered that MITF exists as a hyper-dynamic dimer that is distinct from other general bHLH-Lz transcription factors. Mutations on the dimer interface are deleterious for MITF binding to DNA and its transcriptional activity. These studies revealed a unique opportunity for inhibiting MITF by a direct chemical perturbation of its homodimer. Towards this direction, we have developed a high-throughput screening (HTS)-compatible primary assay that specifically measures the dimerization of MITF in vitro. A screen of the LOPAC has shown a significantly high signal/background window, reproducibility, and a Z' of 0.81. Importantly, the proof-of-principle studies using 3K trial screening have yielded multiple MITF specific disruptors and, their cell permeable derivatives could inhibit MITF transcription activity, pigment formation of primary melanocyte cells and melanoma cell growth with ?M potency. Our central goal team are to implement these HTS-ready assays into a full HTS-campaign using the Scripps Institutional Drug Discovery Library (SDDL) to develop, validate and optimize small molecule inhibitors of MITF. As outlined in Aim 1, we will miniaturize this assay to the 1,536-well plate format, screen a pilot set of 10,000 representative compounds from the Scripps Institutional Drug Discovery Library (SDDL), and use this screen to validate the overall screening process as well as gauge hit rate. Once the HTS automation criteria are met, a full-deck HTS-campaign will be carried out by screening the diverse SDDL library of >640,000 compounds in a tiered approach to discover compounds in Aim 2. In Aim 3, we will use a cascade of follow-up assays to validate screening hits, improve their potency and selectivity, and evaluate their mechanism of action and efficacy both in vitro and in vivo. Collectively, our biochemical and cell-based assays, along with profiling lead compounds against a series of mechanistic screens will drive the discovery of MITF novel and selective molecular modulators for cancer treatment.